Chemistry of bifunctional photoprobes. 4. Synthesis of the chromogenic, cleavable, water soluble, and heterobifunctional sulfosuccinimidyl (N- methylamino perfluoroaryl azido benzamido)-ethyl-1,3'-Dithiopropionate: An efficient protein cross-linking agent

Article abstract of DOI:10.1006/bioo.1998.1098

Synthesis of a new photo cross-linking agent incorporating chromogenicity, cleavability, and water solubility is described. The high efficiency of nitrene insertion observed upon photolysis of perfluoroaryl azides into organic solvents and proteins is extended to the design and synthesis of new multifunctional cross-linking agents useful for protein- protein interactions. The new cross-linker sulfosuccinimidyl (perfluorobenzamido)-ethyl-1,3'-dithiopropionate (SFAD) 10 was conjugated to IgG and cross-linked to horseradish peroxidase (HRP). The analysis of the cross-linked product using ELISA assays leads to a higher yield of IgG-HRP cross-linked product than that seen with a similar nonfluorinated analog. The efficiency of photo cross-linking by SFAD is also extended to small molecule biotin via CH insertion and checked for the retention of binding affinity of the cross-linked product.

Full text of DOI:10.1006/bioo.1998.1098

BIOORGANIC CHEMISTRY 26, 201–212 (1998)
ARTICLE NO. BH981098
Chemistry of Bifunctional Photoprobes1
4. Synthesis of the Chromogenic, Cleavable, Water Soluble, and
Heterobifunctional Sulfosuccinimidyl (N-methylamino Perfluoroaryl
Azido Benzamido)-ethyl-1,3Ј-Dithiopropionate:
An Efficient Protein Cross-Linking Agent
Raghoottama S. Pandurangi*,†, Przemyslaw Lusiak,* Surbhi Desai,‡
and Robert R. Kuntz*
*Department of Chemistry and †Department of Internal Medicine, University of Missouri, Columbia,
65211; and ‡Protein Characterization Group, Pierce Chemical Co., 3747 North Meridian Road,
Rockford, Illinois 61105
Received March 30, 1998
Synthesis of a new photo cross-linking agent incorporating chromogenicity, cleavability,
and water solubility is described. The high efficiency of nitrene insertion observed upon
photolysis of perfluoroaryl azides into organic solvents and proteins is extended to the design
and synthesis of new multifunctional cross-linking agents useful for protein–protein
interactions. The new cross-linker sulfosuccinimidyl (perfluorobenzamido)-ethyl-1,3Ј-
dithiopropionate (SFAD) 10 was conjugated to IgG and cross-linked to horseradish peroxidase
(HRP). The analysis of the cross-linked product using ELISA assays leads to a higher yield
of IgG–HRP cross-linked product than that seen with a similar nonfluorinated analog. The
efficiency of photo cross-linking by SFAD is also extended to small molecule biotin via CH
insertion and checked for the retention of binding affinity of the cross-linked product.
1998
Academic Press
INTRODUCTION
The potential utility of photo cross-linking reagents in the topological mapping
of the binding sites of receptors requires careful consideration of several design
factors in the synthetic steps and an understanding of the productive vs nonproduc-
tive avenues for photoprecursors (2, 3). The combination of chemical and photo-
chemical activation for cross-linking two different proteins is a powerful procedure
for identifying the specific amino acids involved within the protein–protein binding
sites. Protein photo cross-linking agents typically contain a chemical functional
group (e.g., succinimide, sulfosuccinimide, NCS, etc.) for nucleophilic substitution
on the first protein, a disulfide bond, and a photochemically active group which
remains chemically inert until photoactivation in the presence of a second protein
(4). Following irradiation and photochemically induced covalent linking with the
1 For part 3, see Ref. (1).
2 To whom correspondence should be addressed at 601S, College Avenue, 123, Chemistry Department,
University of Missouri, Columbia, MO 65211. Fax: (573) 882 2754. E-mail: raghu@missouri.edu.
201
0045-2068/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.

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PANDURANGI ET AL.
second protein, cleavage of the disulfide (S–S) bridge of the cross-linked protein
results in the concomitant transfer of the marker (e.g., radioactivity or chromogen-
icity or fluorescence) to the specific amino acid(s) at which the cross-link occurs.
Hence, a useful photo cross-linker should have a marker and be water soluble and
cleavable under mild conditions (5–8). Introduction of additional features like
fluorescence, chromogenicity, or provision for attachment of a radioactive marker
may enhance the utility of cross-linking agents. However, the efficiency of photocon-
jugation and the stability of the covalent bond formed by the photoprecursors
under the conditions used for the analysis of the cross-linked products essentially
determine the final yield of the cross-linked conjugate (9). A high efficiency of
photoconjugation is necessary for the recovery of the cross-linked product in suffi-
cient yield for characterization by macroscopic techniques.
Study of the fundamental photochemistry of perfluoroaryl azides by Keana et al.
(10, 11) and the development of high efficiency heterobifunctional perfluoroaryl
azide based chelating agents for covalent attachment to proteins and antibodies in
our laboratory (12–16) and by others (17, 18) suggest that perfluoroaryl azides
are potentially good candidates as cross-linking agents. The promising nature of
perfluoroaryl azides as cross-linkers stems from the fact that singlet perfluoroaryl
nitrene rearranges more slowly to didehydroazipines, allowing the reactive interme-
diate to react with even unactivated CH bonds by formal insertion (19–23). Recently,
we have shown from flash photolysis studies that the bifunctional nitrenes carrying
electron withdrawing groups (amide and ester groups) exhibited a longer lifetime
for singlet nitrenes (Ͼ250 ns), leading to the highest CH insertion in model solvents
(1) and in proteins (12, 15, 16). Such observations prompted us to design and report,
here, a first example of the synthesis of a chromogenic, cleavable, water soluble, and
heterobifunctional sulfosuccinimidyl (N-methylamino perfluoroazido benzamido)-
ethyl-1,3Ј-dithiopropionate 7 (Scheme 1). We also wish to report the preparation
of sulfosuccinimidyl (perfluoroaryl azido benzamido)-ethyl-1,3Ј-dithiopropionate
10 (Scheme 2) and comparison of its relative cross-linking efficiency with the com-
mercially available sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1,3Ј-dithiopro-
pionate 11 (SASD) cross-linker. We have extended our studies to cross link small
molecules and demonstrate the retention of native characteristics of biotin (e.g.,
binding affinity toward streptavidin) in the post-cross-linking stage.

CHEMISTRY OF BIFUNCTIONAL PHOTOPROBES
203
EXPERIMENTAL
All synthetic procedures were conducted in a dry nitrogen atmosphere using
standard Schlenk tube techniques and prepurified solvents. Reactions involving the
synthesis of azides were carried out under subdued light by wrapping the flasks
with aluminum foil. Nuclear magnetic resonance spectra were recorded in CDCl3
on a Bruker 250 MHz spectrometer and chemical shifts are reported in ppm down-
1
field from SiMe4 for H NMR. 19F NMR chemical shifts are reported with respect
to CFCl3 as an external standard. Infrared spectra were recorded as KBr pellets
on a Nicolet 20DXB FT-IR spectrophotometer. Elemental analysis for the new
compounds were performed by Oneida Research Services, Inc. (New York). UV/
visible spectra were recorded either in water or in buffer (0.1 M sodium phosphate,
SCHEME 1

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PANDURANGI ET AL.
SCHEME 2
150 mM NaCl, pH 7.4) on a Hewlett Packard (8452A) diode array spectrophotome-
ter. Photolyses were carried out with a 200-W high-pressure Hg lamp with water
as a filter. Compounds 5 (24) and 8 (10) were prepared by literature methods. The
nonfluorinated cross-linker 11 is available commercially from Pierce Co.
Synthesis of 2
A solution of methyl-4-azidotetrafluorobenzoate 1 (1.97 g, 7.91 mmol), prepared
as reported earlier (10), MeNH2 · HCl (1.33 g, 19.78 mmol), and 8 ml Et3N in 10
ml acetonitrile was refluxed for 18 h. The solution was cooled in ice and Et3N · HCl
was filtered and the solvent was evaporated in vacuum. The crude product was
washed with NaHCO3 solution and extracted into CH2Cl2 and dried using MgSO4.
The filtrate was evaporated to leave methyl 4-azido-N-methyl-2-amino trifluoro-
1
benzoate 2 (1.696 g, 51.6%). H NMR (CDCl3), ͳ, 7.00, (br, s, 1H), 3.91, (s, 3H),
3.06, (m, 3H). 19F NMR (CDCl3), ͳ, Ϫ135.47 (1F), Ϫ147.25 (1F), Ϫ163.95 (1F).
Synthesis of 3
A solution of methyl 4-azido-N-methyl-2-amino trifluorobenzoate 2 (583 mg, 2.24
mmol) in methanol was stirred overnight with 2 ml 30% aqueous NaOH at RT.
The solution was acidified by 2 N HCl in an ice bath to pH Ͻ1 and extracted by
CHCl3. The extract was dried using MgSO4 and the solvent was evaporated to
leave 4-azido-N-methyl-2-aminotrifluorobenzoic acid 3 (523.79 mg, 95%). 1H NMR
(DMSO-d6), ͳ, 3.75, (br, s, 1H), 3.89, (d, 3H). 19F NMR (DMSO-d6), ͳ, Ϫ134.48
(1F), Ϫ146.95 (1F), Ϫ163.71 (1F).

CHEMISTRY OF BIFUNCTIONAL PHOTOPROBES
205
Synthesis of 4
The carboxylic group of 4-azido-N-methyl-2-aminotrifluorobenzoic acid 3 in dry
CH2Cl2 (246.14 mg, 1 mmol), was activated by N-hydroxysuccinimide (NHS, 115
mg, 1 mmol), in the presence of dicyclohexylcarbodiimide (DCC, 220 mg, 1.1 mmol),
also taken in CH2Cl2 (10 ml) and stirred at RT overnight. The mixture was
filtered. The filtrate was evaporated to obtain of N-succimidyl-4-azido-2,3,5,6-tetra-
1
fluorobenzoate 4 (340 mg, 99%). H NMR (CDCl3), ͳ, 3.16, (m, 3H), 2.98, (s, 4H).
19F NMR (CDCl3), ͳ, Ϫ130.97 (1F), Ϫ146.84 (1F), Ϫ163.39 (1F).
Synthesis of 6
A mixture of 3-[(2-aminoethyl)dithio]propionic acid in 1ml of water (78 mg, 0.43
mmol), sodium bicarbonate (72 mg, 0.86 mmol) was added to a solution of
N-succimidyl-4-azido-N-methyl-2-amino-3,5,6-trifluorobenzoate 4 (147.6 mg, 0.43
mmol) taken in 14 ml dioxane. After stirring the mixture for 18 h, the solvent was
evaporated and reduced to 2 ml volume, cooled in ice, and adjusted to pH 2 with
concentrated HCl to yield the solid product (79.2 mg, 45%) which was directly used
1
for conjugating sodium salt of sulfosuccinimide. H NMR (CDCl3), ͳ, 7.75, (b, s,
1H), 6.89, (br, m, 1H), 3.12 (m, 3H), 2.93–1.9 (m, 8H). 19F NMR (CDCl3), ͳ, Ϫ141.94
(1F), Ϫ146.04 (1F), Ϫ163.87 (1F).
Synthesis of 7
A mixture of 6 (135 mg, 0.33 mmol), sodium salt of N-hydroxysuccinic acid (71
mg, 0.33 mmol), and DCC (72 mg, 0.35 mmol) was dissolved in 2 ml DMF and
stirred at room temperature overnight. The reaction mixture was cooled and the
precipitated dicyclohexylurea was removed by filtration. The filtrate was washed
with a small quantity of dry DMF. The required product 7 was then precipitated
from the solution by addition of ȁ20 vol of ethyl acetate, filtered, and dried in high
1
vacuum. H NMR (DMSO-d6), ͳ, 8.81 (b, 1H), 5.61, (b, 1H), 3.95 (m, 1H), 3.21
(m, 3H), 3.01–2.6 (m, 10H). 19F NMR (DMSO-d6), ͳ, Ϫ143.95 (m, 1F), Ϫ146.97
(m, 1F), Ϫ164.51 (m, 1F). UV (water), ␭
356 nm. Anal. Calcd for
max
C17H16N6F3O8S3Na: C, 33.56; H, 2.65; N, 13.81. Found: C, 33.12, H, 2.55; N, 13.45.
Synthesis of 9
A mixture of 3-[(2-aminoethyl)dithio]propionic acid in 1 ml of water (78 mg 0.43
mmol), sodium bicarbonate (72 mg, 0.86 mmol) was added to a solution of
N-succimidyl-4-azido-2,3,5,6-tetrafluorobenzoate prepared as reported earlier (10)
(149.5 mg, 0.45 mmol) taken in 5 ml dioxane. After stirring the mixture for 18 h,
the solvent was evaporated partially to 2 ml volume, cooled in ice, and adjusted to
pH 2 with concentrated HCl to yield the solid product (68.1 mg, 38% yield) which
1
was directly used for conjugating sulfosuccinimide. H NMR (CDCl3/DMSO-d6),
ͳ, 8.85, (b, s, 1H), 2.9–1.8 (m, 8H) 19F NMR (CDCl3/DMSO-d6), ͳ, Ϫ141.57 (2F),
Ϫ151.44 (2F).

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PANDURANGI ET AL.
Synthesis of 10
A mixture of 9 (50 mg, 0.126 mmol), the sodium salt of N-hydroxysuccinic acid
(27 mg, 0.13 mmol), and DCC (30 mg, 0.17 mmol) was dissolved in 2 ml DMF and
stirred at room temperature overnight. The reaction mixture was cooled and the
precipitated dicyclohexylurea was removed by filtration. The product 10 was then
reprecipitated from the solution by addition of ȁ20 vol of ethyl acetate, filtered,
1
and dried in high vacuum. H NMR (DMSO-d6), 8.31 (b, s, 1H), 3.85 (m, 1H),
2.9–1.8 (10H). 19F NMR (DMSO-d6), ͳ, Ϫ135.54 (2F), Ϫ151.30 (2F). UV (Water),
␭max 275 nm, 310 nm(sh). Anal. Calcd for C16H12N5F4O8S3Na: C, 32.17; H, 2.02; N,
11.72. Found: C, 32.27; H, 2.12; N, 11.82.
Biological Assay
All the following steps were carried out under diffused light. The protocol for
conjugation of cross-linkers SASD (10 Ȑl) and 10 [10 Ȑl, made up at 1 mg/ml] in
PBS, (0.1 M sodium phosphate, 150 mM NaCl, pH 7.4)] involved incubation of
each one of them separately with 2 mg mouse IgG (at 11 mg/ml) in the dark for
2 h. Samples were injected into 0.1–0.5 ml Slide-A-Lyzers dialysis cassettes (Pierce)
and dialyzed overnight versus 5 L of PBS. The samples were removed from the
dialysis cassettes and placed in 16 ϫ 100-mm glass tubes. Horseradish peroxidase
(HRP, Pierce, 10 mg) was dissolved in 1 ml PBS and 200 Ȑl of the HRP was added
to the activated mouse IgG. A hand-held long wavelength UV light was placed
above the samples and the samples were irradiated with UV light for 30 min before
storing them at 4ЊC.
The analysis of the conjugates was done using the ELISA assay. The two mouse
IgG–HRP conjugates and HRP (10 mg/ml, diluted with 190 Ȑl of PBS) were further
diluted (1 : 100) with PBS. 100 Ȑl of each dilution was added to separate wells (the
assay was performed in duplicate) of a goat anti-mouse IgG coated microtiter plate.
The last row was a PBS blank with no conjugate or HRP. Prior to the addition of
the sample, the plate had been washed with 3 ϫ 200 Ȑl PBS. The samples were
incubated in the plate wells for 1 h at room temperature. The plate was washed
again with 3 ϫ 200 Ȑl PBS. Turbo TMB (tetramethyl benzidine, 100 Ȑl) was added
to each well. The plate was incubated for 5 min at room temperature before adding
1 M H2SO4 to each well to stop the reaction. Subsequently, the plates were read
at 450 nm for optical densities.
Biotinylation of Rabbit IgG
About 1 mg of rabbit IgG in 90 Ȑl of PBS was added to the solution of SFAD at 12
mg/ml and the reaction was monitored for 2 h at room temperature. The sample was
desalted using 5 ml cross-linked Dextran column (Pierce) before adding 2 ml of 2 mM
biotin. The sample was exposed to UV light at 312 nm for 30 min. A goat anti-rabbit
coated plate was washed (3 ϫ 20 ml) with PBS/0.05% Tween 20 (Pierce). Biotinylated
rabbit IgG was serially diluted (1 : 1) and incubated for 1 h. The plates were washed
again before adding streptavidine–HRP (1 mg/ml, Pierce). Quantification of IgG
cross-linked product was determined by adding TMB substrate to each incubation

CHEMISTRY OF BIFUNCTIONAL PHOTOPROBES
207
well and the reaction was monitored for 10 min before stopping with 1 M H2SO4 and
optical density at 4560 nm was noted using a microwell plate reader.
RESULTS AND DISCUSSION
Chromogenic nitrene precursors which are red shifted from the protein absorption
maximum have been shown to be versatile photolabeling agents for human serum
albumin (HSA) since these precursors have an additional advantage of absorption
at long wavelength (15). Nucleophilic substitution of 4-azido perfluoro methyl ben-
zoate 1 by methyl amine hydrochloride leads to the N-methyl trifluoro derivative
2 selectively (Scheme 1). The interesting aspect of this nucleophilic substitution is
a formation of chromogenic derivative 2 shifting ␭max to 356 nm, far away from the
␭
max
absorption of biomolecules. Hydrolysis of 2 by 30% NaOH gives rise to the
corresponding acid 3 which can be activated to the corresponding succinimide 4
using DCC. The introduction of an S–S cleavable bond is achieved by reaction of
4 with 3-[(2-aminoethyl)dithio]propionic acid 5, prepared by a method found in
the literature (24). In the last step, activation of the carboxylic group by the sodium
salt of sulfosuccinimide in DMF lead to a chromogenic, cleavable, water soluble,
and bifunctional cross-linker 7. The new cross-linker was characterized by
multiprobe NMR (1H, 13C, and 19F), IR and elemental analysis. To illustrate the
relative effectiveness of the perfluorinated aryl azide photoprobe 10, a structural
analog of SASD 11 was prepared and used in comparative cross-linking studies.
The biological protocol for cross-linking mouse IgG to the enzyme is outlined in
Fig. 1. The procedure involved (a) conjugation of the cross-linker with mouse IgG
through the interaction between the sulfosuccinimide group of the cross-linker and
Ͱ- or ␧-amino groups of IgG, followed by a separation of the unconjugated cross-
linker by extensive dialysis, and (b) irradiation of the conjugated mouse IgG in the
presence of HRP using UV light. Analysis of the cross-linked product was carried
out using the ELISA assay. The mouse IgG–HRP conjugate was added and serially
diluted (1 : 1) in wells coated with goat anti-mouse IgG. The plate was washed with
phosphate buffer (PBS) containing 0.05% Tween 20 to remove any HRP that was not
conjugated with mouse IgG. The amount of HRP associated with mouse IgG was
estimated from the enzyme immunoassay using 3, 3Ј,5,5Ј-tetramethylbenzidine
(TMB) as a substrate which allows a detection of HRP (12). The absorbance of the
enzyme-substrate (OD) is proportional to the concentration of HRP bound to mouse
IgG (i.e., cross-linked to antibody). Employing the identical conditions for the SASD
cross-linking assay permits a comparison of the cross-linking efficiencies of 10 and
SASD. PBS buffer with no conjugate or HRP forms the control.
The results on the comparison of cross-linking efficiencies between 10 and SASD
11 is shown in Fig. 2. The nature of the saturation of optical density followed by
a slight increase for nonfluorinated analog 11 is surprising. Although, at present
we do not know the explanation for this behavior, at higher concentrations
(50 Ȑg/ml) the data indicate that the conjugate made with the perfluoroaryl function-
alized cross-linker 10 gave 40% higher OD compared to SASD. This can be attrib-
uted to the higher incorporation of HRP cross-linking to mouse IgG. The typical

208
PANDURANGI ET AL.
FIG. 1. Cross-linking protocol for compound 10 with IgG followed by irradiation with HRP.
higher photochemical conjugation efficiency of perfluoroaryl azides reported in
model solvents (21) and proteins (12), for example, could, in principle, explain the
superiority of perfluoroaryl based cross-linkers compared to the nonfluorinated aryl
azide analog which are known to undergo ring expansion rather than the formal
insertion of nitrene. Conventional photolabile cross-linking agents involving aryl
azides with 125I markers suffer from low insertion efficiency since they either re-
arrange to didehydroazipines or convert to triplet nitrene intermediates leading to
noninsertion polar product (25). In addition, the release of iodine radiolabel into
the surrounding macromolecules in a nonspecific manner during photolysis results in
the labeling of macromolecular domains other than those involved in ligand–affinity
interactions (26). Moreover, it has been established that the presence of a heavy
atom such as iodine in the same ring as the azide functionality reduces the reactivity
of the nitrene leading to triplet derived products through intersystem crossing (ISC)
rather than the desired singlet derived insertion products (27, 28). Extensive studies

CHEMISTRY OF BIFUNCTIONAL PHOTOPROBES
209
FIG. 2. Relative cross-linking efficiencies of compounds 10 and 11 with IgG–HRP conjugate.
on the photochemistry of phenyl azides by Schuster et al. (19, 20) and Platz et
al. (21–23) reveals the limited success of aryl azides as cross-linkers. Although
didehydroazipines (photolytic product from aryl azides) are capable of adding to
nucleophiles, they might be of limited use in cross-linking studies since they are
less reactive than nitrenes and are not stable under the proteolytic conditions
typically used for peptide mapping and amino acid identification, thus leading to
a low yield of the cross-linked product. On the other hand, moderately successful
labeling reactions with azides (e.g., azido nucleotides (29)) may be attributed to
the preassociation of the probe to a particular region of the macromolecule to be
labeled where the generated reactive nitrene is restricted and not allowed to diffuse
through the medium and, hence, reacts within the immediate vicinity leading to
insertion. Goeldner et al. (30) have established a hierarchy between nonflourinated,
diflourinated, and tetrafluorinated aryl azido probes which supports the potential
application of perfluoroaryl based reagents for efficient labeling, for example of
GABA receptors. These results also agree with the reports on a similar nonfluori-
nated cross-linker, SAED, which shows only 15% cross-linking efficiency (31).
In the area of cross-linking of proteins, it is important to demonstrate the retention
of the native characteristics of biomolecules such as binding affinity in the post-

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PANDURANGI ET AL.
FIG. 3. Binding data on IgG–SFDA biotin conjugate.
cross-linking stage. In this connection, we have photoconjugated IgG–SFAD to
biotin which is known to bind streptavidin strongly (32). The choice of biotin is
based on the establishment of the binding protocol of biotin with streptavidin–HRP,
which can be detected and quantified easily. The protocol involves conjugation of
IgG with SFAD followed by irradiation with biotin. Binding of biotinylated IgG–
SFDP with goat anti-rabbit IgG coated plate followed by repeated wash with buffer
gives rise to pure IgG-SFDP cross-linked to biotin. The binding assays involved
incubation of the cross-linked product with streptavidin–HRP and monitored
through OD at 450 nm after treating the assay with TMB, followed by the addition
of 1 M H2SO4. Figure 3 shows the binding data for biotinylated IgG–SFAD cross-
linker indicating a high binding affinity even at 0.75 mg concentration and showing
saturation binding at higher concentrations. These data suggest that protocol
adopted for cross-linking biomolecules using SFAD does not interfere with the
binding regions and hence, retains the native characteristics of biomolecules. The
retention of the binding affinity of biotin can be rationalized in terms of CH insertion
of singlet perfluoroaryl nitrene on the hydrophobic part of biotin. In an alternate
experiment involving the photo cross-linking in the presence of a triplet sensitizer
(e.g., acetophenone), no binding of IgG–SFAD to biotin was observed, indicating
that the triplet perfluoroaryl nitrene produced an aniline type of product which
cannot form covalent linkage with biotin, unlike CH insertion by singlet nitrene.
CONCLUSIONS
The main consideration for the successful application of covalent attachment of
photolabels to biomolecules is the evaluation of their bimolecular insertion efficien-
cies in buffer conditions. We have introduced chromogenicity (bathochromic shift)

CHEMISTRY OF BIFUNCTIONAL PHOTOPROBES
211
and cleavability into the new design of perfluoroaryl functionalized cross-linker
based on our earlier studies on the high efficiency of nitrene insertion into HSA,
IgG, and B72.3 antibody by functionalized perfluoroaryl azides (12–15). The new
photo cross-linker is superior to the nonfluorinated analog, demonstrating 40%
higher incorporation into HRP. The technical superiority is correlated well with
the differential nature of the reactive intermediates involved and singlet lifetimes
between perfluoroaryl azides and conventional aryl azides. The higher efficiency
of cross-linking, shown by perfluoroaryl derivatives with a cleavable arm, permits
the tranfer of perfluoraryl group to the target protein which can be monitored by
19F NMR and, hence, may find use in better characterization of the binding sites
involved in protein–protein cross-linking by macroscopic techniques. Binding stud-
ies with biotin by preconjugated IgG–SFAD demonstrates the retention of the
binding affinity in the post-cross-linking stage enhancing their utility. Further studies
on the peptide mapping of the cross-linked product are in progress and will be
published elsewhere.
ACKNOWLEDGMENTS
The authors thank Dr. Robert Vigna (Pierce) for providing useful bioconjugation and cross-linking
data on our cross-linker. We also acknowledge the useful discussions with Prof. Krishna Sharma,
Ophthalmology Department, University of Missouri. We thank Prof. Wynn Volkert for the partial finan-
cial support to Dr. P. Lusiak.
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